There is a fundamental gap in the understanding of molecular mechanism involved in regulating lytic DNA replication of Kaposi's sarcoma associated herpesvirus (KSHV) also known as Human Herpesvirus 8 (HHV8). Lytic DNA replication is important in inducing KSHV mediated tumorigenesis, evidenced by the fact that AIDS patients receiving anti-herpetic drugs, Ganciclovir (GCV), Fascarnet (PFA) and N- methanocarbathymidine, which blocks lytic DNA replication, had reduced risk for Kaposi's sarcoma (KS). KSHV is tightly linked to various human malignancies including Primary Effusion Lymphomas (PELs), Kaposi's sarcoma (KS) and Multicentric Castleman's Disease (MCDs) and cause tumors predominantly in immune compromised individuals including HIV infected individuals and patients receiving immune suppressive therapies to prevent graft rejection. KSHV induced tumors are one of the major causes of morbidity and mortality of HIV/AIDS patients. This study is important because the incidence of immunosuppression is still a concern due to HIV infection and the use of immunosuppressive drugs in organ transplant/autoimmune patients. Introduction of HAART (highly active anti-retroviral therapy) have reduced the incidence of KS tumors but there are no specific treatment for KS tumors. The long-term goal of this project is to define the mechanism of lytic DNA replication and to identify the factors regulating DNA replication, which can be exploited for devising strategies to block KSHV pathogenesis. The objective of this application is to determine the mechanism used by a functional lytic origin and the role of viral exonuclease in genome amplification. This study will also determine the role of viral microRNA and epigenetic modifications in regulating replication initiation and virion production. Our preliminary data shows that single origin of replication (oriLyt-L) is preferentially used for initiating replication in a bidirectional manner. Our central hypothesis is that replication initiations at the lytic origins are regulated by genetic and epigenetic factors. The hypothesis has been formulated based on the preliminary data produced in our laboratory on replication initiation using biochemical and genetic approaches. The rationale for the proposed research is that determining molecular mechanism of lytic DNA replication will provide potential therapeutic avenues for thwarting the virus from infected cells o treat KSHV associated malignancies, which are a major health problems for HIV infected and organ transplant patients undergoing immunosuppressive therapies to prevent graft rejection. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) Identification of the functional replication origin and the mechanism of lytic DNA replication using SMARD, 2) Determining the control mechanism of oriLyt-mediated replication, and 3) Determining the dynamics of epigenetic modifications at oriLyt, which may dictate origin firing. Under the first aim, an already established approach, single molecular analysis of the replicated DNA (SMARD), will determine preferentially used replication initiation origin for viral genome replication in various cell backgrounds. Role of viral exonuclease will also be determined in generating unit length viral genome for packaging. Under the second aim, role of viral microRNA, binding to targets or transcription of its pre-miRNA through lytic origin, will be evaluated for replication initiation. Under the third aim, epigenetic modifications of chromatins a lytic origins will be investigated for their contribution in controlling origin usage. The approachis innovative, because we are using a powerful in-cell labeling of replicating DNA procedure, SMARD to determine origin usage, replication fork progression and branching in DNA after replication. We are also using innovative galK-Kan procedure to introduce point mutations in KSHV genes for determining specific protein functions to better understand their roles in the context of virus. The proposed research is significant, because it is expected to vertically advance and expand the understanding of lytic DNA replication in amplifying viral genome during viral reactivation. Ultimately, such knowledge could potentially be used for devising interventional strategies to block KSHV induced malignancies.